The recent success in reprogramming somatic cells into induced Pluripotent Stem (iPS) cells by defined factors has opened exciting possibilities not only for the investigation of complex human diseases in the Petri dish but also for the ultimate application in transplantation therapy. Direct reprogramming provides for the first time the opportunity to generate in vitro and in vivo models of complex disorders such as familial and sporadic Parkinson's disease using patient-specific cells. However, major technical hurdles need to be resolved to realize the full potential of the iPS system for the study and eventual therapy of human disease. The experiments proposed in this application are a direct extension of our work with mouse cells and are aimed at establishing robust methods for reprogramming human somatic cells and setting up effective protocols for manipulating human ES (hES) cells and iPS cells. This proposal has 4 aims: 1. We will devise robust approaches to target genes in hES and iPS cells. Gene targeting is crucial for the reprogramming of somatic cells into iPS cells and for differentiating ES cells and iPS cells in vitro into functional cells that could be used for eventual therapy of degenerative disorders. 2. We will seek to elucidate the molecular mechanisms of reprogramming human somatic cells. Our goals include the design of approaches that avoid genetic alteration of the donor cells, to assess whether insertional mutagenesis contributes to iPS cell generation, and to define the molecular signature of different reprogramming stages. 3. We will establish high throughput screens to identify small molecules that increase efficiency of reprogramming and substitute for the need to introduce any of the factors by retrovirus-mediated gene transfer. 4. We will establish protocols that eventually will allow the study of complex human diseases using patient-specific iPS cells both in the Petri dish as well as under in vivo conditions. Finally, we will establish procedures to derive iPS cells from peripheral human blood samples. It is our hope that the experiments proposed in this application will contribute to solving some of the crucial obstacles that presently hamper the application of this technology to the study of human disease and to ultimately use it for transplantation therapy of degenerative disorders. PUBLIC HEALTH RELEVANCE: This grant proposal seeks to define the molecular mechanisms that bring about the conversion of human somatic cells to a pluripotent state, to devise strategies for assessing the developmental potential of human iPS cells, and to achieve reprogramming without the need for genetic manipulation. It is our hope that the experiments proposed in this application will contribute to solving some of the crucial obstacles that presently hamper the application of the technology to the study of human disease and to eventually use the technology for transplantation therapy of degenerative disorders. Once the experimental obstacles have been overcome, it is our long-term goal to use patient-specific iPS cell lines to study the basis of complex human diseases.